ABSTRACT Congenital heart disease, the most common birth defect, is frequently associated with deficient heart muscle, leading to heart failure. Currently, the only way to replace heart muscle cells, cardiomyocytes, is through heart transplantation. Regenerative therapies would transform the treatment of congenital heart disease and save many lives. We study the mechanisms of cardiomyocyte proliferation with the aim of increasing this process therapeutically. We have previously demonstrated that extracellular factors can be used to stimulate cardiomyocyte proliferation, leading to improved myocardial structure and function in animal models of heart failure. The clinical translation of this innovative approach requires understanding of how cardiomyocytes are able to perform two completely different tasks: contraction of myofibrils and cell division. We have shown that during cell division cardiomyocytes divide their contractile apparati, which consist of myofibrils, but the detailed mechanisms are not understood. It has been shown that myofibril formation and cardiomyocyte cytokinesis are controlled by mechanisms involving p38 mitogen-activated protein kinase (MAPK), but the role of p38 in myofibril disassembly remains unknown. Our preliminary data indicate that cardiomyocyte cell cycle activity in humans is highest in infants, suggesting that regenerative cardiomyocyte proliferation may be most effectively stimulated in this age group. We will therefore perform our investigations in neonatal animals. We hypothesize that myofibril disassembly in proliferating neonatal cardiomyocytes is a conserved, multi-step process that is controlled by a mechanism involving p38 MAPK and is associated with brief reduction of cardiomyocyte contractile function. In Aim 1 we will define and characterize the disassembly process. In Aim 2, we will modify p38 signaling and determine the effects on myofibril disassembly. In Aim 3, we will determine the effect of myofibril disassembly on cardiomyocyte function in the intact heart. The results of this research should increase the translational potential of regenerative strategies that stimulate cardiomyocyte proliferation.